Long wavelength ultraviolet light is absorbed by the lens leading to interactions which have been considered both beneficial and deleterious to vision. Central to the hypotheses relating the effects of chronic exposure of the lens to ultraviolet radiation is the role of the indole ring of tryptophan. Furthermore, superimposed on the normal alteration of lens proteins with aging, the oxidation of sulphur containing amino acid residues is strikingly correlated with progressive cataract formation. The main aim of this project is to understand at the atomic level the twin effects of photo-excitation and oxidation on the interactions of a lens protein which occurs predominantly in the lens nuclear region. Using the techniques of X-ray diffraction, this laboratory has discovered that the disposition of sulphur and aromatic residues in Gamma-crystallin II displays a spatial arrangement which suggests they may cooperate in providing molecular stability. The particular objective of this proposal is to determine, by using a variety of photo-oxidative challenges on crystals of Gamma-II, the influence of the oxidative state of the protein on the photo-destruction of aromatic amino acids. This will be done by extending the very high resolution diffraction data set of Gamma-II by oscillation camera photography using a synchrotron X-ray radiation source. This will be followed by data processing and refinement of the current 1.6 angstroms structure to atomic resolution. Then a structure determination will be done using crystals which have been specifically recorded at sub-zero temperatures in order to preserve the uv-modified crystal. The data recorded will result in calculation of electron density difference maps. Separate crystals will subsequently be subjected to various combinations of photo-oxidative treatments, resulting in electron density maps of the different stages. The sites of extra oxygen atoms will be seen from which mechanistic pathways can be derived. The main burden of this proposal is the request for financial support for Dr. Huub Driessen to carry out this programme. The long term objectives reside in that once the biophysics of the normal interactions are understood, and clues to the chemistry of photo-oxidation gained by a combination of crystallographic techniques from this laboratory and biochemical and spectroscopic studies of other laboratories, then a rational approach can be implemented to the design of inhibitors of harmful processes related to photo-oxidation.